Are xanthosine and 7-methylxanthosine caffeine precursors?

Although caffeine biosynthesis has been reported to start with the methylation of xanthosine to yield 7-methylxanthosine, we failed to demonstrate in suspension-cultured cells of coffee the presence of 7-methylxanthosine, even after considerable stimulation of overall purine alkaloid (PA) formation by means of ethephon or/and adenine. Hence, in order to increase sensitivity and to study incorporation kinetics, stimulation was combined in the present work with radioactive labelling; [14C] adenine or [methyl-14C] methionine was fed to cells with PA synthesis enhanced either by 5 mM ethephon or by 1 mM adenine. Ethephon treatment resulted in a high (50 to 80% after 24 hr) incorporation of adenine radioactivity into PA, whereas pre-incubation with 'cold' adenine known to increase long-term PA formation strongly reduced that incorporation in favour of labelling xanthosine and related nucleosides. Neither ethephon nor adenine treatment led to a distinct incorporation of [14C]adenine or [methyl-14C]methionine into 7-methylxanthosine. Traces of [14C]7-methylxanthosine (less than 1%) could only be detected in cells pre-incubated with 1 mM adenine and thus exhibiting 'artificially' enlarged endogenous xanthosine pools either strongly labelled (after [14C]adenine-feeding) or unlabelled (after [methyl-14C]methionine-feeding). Kinetic analyses upon labelling with [14C]adenine revealed that, based on the specific radioactivity, xanthosine must be excluded as a precursor in caffeine biosynthesis, unless we consider separate pools of xanthosine, one being a collecting pool, the other the PA biosynthesis pool. Therefore, if we adhere to the xanthosine hypothesis, we should postulate metabolic channelling of the early events in PA biosynthesis, including formation of xanthosine, its methylation to 7-methylxanthosine, and finally enzymatic hydrolysis to 7-methylxanthine. However, currently there is no argument against the possibility of the first methylation taking place at the nucleotide level.